Redalyc.Patterns of Leaf Epicuticular Waxes in Species of Clusia

Interciencia ISSN: 0378-1844 [email protected] Asociación Interciencia Venezuela

Medina, Ernesto; Aguiar, Guillermina; Gómez, Matilde; Medina, José D. Patterns of leaf epicuticular waxes in species of clusia: taxonomical implications Interciencia, vol. 29, núm. 10, octubre, 2004, pp. 579-582 Asociación Interciencia Caracas, Venezuela

Available in: http://www.redalyc.org/articulo.oa?id=33909707

How to cite Complete issue Scientific Information System More information about this article Network of Scientific Journals from Latin America, the Caribbean, Spain and Portugal Journal's homepage in redalyc.org Non-profit academic project, developed under the open access initiative PATTERNS OF LEAF EPICUTICULAR WAXES IN SPECIES OF Clusia: TAXONOMICAL IMPLICATIONS

Ernesto Medina, Guillermina Aguiar, Matilde Gómez and José D. Medina

SUMMARY

The genus Clusia L. (Clusiaceae) encompasses ca. 300 species quantity of hexane-soluble compounds extractable from the leaf sur- and occurs from southern USA and Mexico, to southern Brazil and face, which amount to >90% in Clusia rosea, C. orthoneura, and C. Bolivia. It includes free-standing trees and shrubs, hemiepiphytes, minor, the presence of the triterpenes α-amyrin and lupeol in C. epiphytes, and lianas. Taxonomic analysis of this genus is difficult multiflora, and of friedelin and taraxerol, together with C33 and because of the poor preservation of floral material after drying. C35, in C. grandiflora and C. schomburgkiana. The results suggest This work explores the composition of epicuticular waxes in order that the relative proportions of alkanes and triterpenoids in epicu- to allow characterization, at the species level, using chemical mark- ticular waxes may have taxonomic significance for separating spe- ers. The six species analyzed could be separated using the relative cies or infrageneric sections.

RESUMEN

El género Clusia L. (Clusiaceae) comprende unas 300 Las especies pudieron separarse en base a la proporción de especies que ocurren desde México y el sur de EEUU hasta alcanos, >90% del total en Clusia rosea, C. orthoneura y C. mi- Bolivia y el sur de Brasil. Entre ellas se incluyen árboles y nor, a la presencia de los triterpenos α-amirina y lupeol en C. arbustos, hemiepifitas, epifitas y lianas. El análisis taxonómico multiflora, y de friedelina y taraxerol, conjuntamente con C33 y del género se dificulta por la pobre preservación de las flores al C35 en C. grandiflora y C. schomburgkiana. Los resultados ser secadas. Este trabajo explora la composición de ceras sugieren que la proporción de alcanos y triterpenoides de ceras epicuticulares para caracterizar especies mediante marcadores epicuticulares tiene importancia taxonómica y puede ser utiliza- químicos. Se analizó la composición del extracto obtenido de da para separar especies o secciones infragenéricas. seis especies mediante lavado de la superficie foliar con hexano.

Introduction Clusia species possess charac- (Bittrich and Amaral, 1996; etration of liquid water into teristics possibly associated Marsaioli et al., 1999). intercellular spaces, and The genus Clusia includes with tolerance to dry condi- Epicuticular waxes play a avoiding the establishment of around 300 species that occur tions (succulent and/or leathery role in plant biology in water epiphyllic organisms (Nein- throughout the interneotropi- leaves, low leaf conductance; loss regulation, because cu- huis and Barthlott, 1997). cal realm, from southern USA Lüttge, 1996; Pipoly et al., ticular transpiration is related The composition of epicu- and Mexico to southern Brazil 1998). Several species have to the permeability of the cuticular waxes is dominated by and Bolivia (Pipoly et al., been shown to be constitutive ticle to water vapor and to long- (>C21), frequently odd- 1998). It includes trees and CAM, or switching from C3 the habitat in which plants chain alkanes (Barthlott, shrubs that grow as free- to CAM metabolism under grow (Schreiber and Riederer, 1989). Other related com- standing individuals on a vari- drought conditions (Franco et 1996). Shady and/or humid pounds are very long chain ety of substrates (shallow clay al., 1990; Lüttge, 1996). habitats frequently produce alcohols and acids, and in or deep sandy soils, calcare- Many Clusia species pro- leaves more permeable to wa- many species triterpenoids ap- ous or serpentinitic soils), or duce resiniferous waxes in the ter vapor than leaves from pear in amounts usually as hemiepiphytes, epiphytes staminate, and/or the pistillate dry and/or sunny exposed small. The amount of epicu- and lianas. flowers, providing material for habitats (Bondada et al., ticular waxes in a given plant All species produce latex honey bees nest construction 1996). In addition, epicuticu- population may be used to varying in abundance, density, (Cuesta-Rubio, 2002). Produc- lar waxes act as insulators identify ecological conditions and color (Engler, 1925; tion of these resins is signifi- from excess environmental (drought and sun exposure), Pipoly et al., 1998). Numerous cant as a pollinator attractant humidity, preventing the pen- while its composition may

KEYWORDS / Alkanes / Chemotaxonomy / Clusia / Epicuticular Waxes / Triterpenes / Received: 04/12/2004. Modified: 08/23/2004. Accepted: 08/26/2004.

Ernesto Medina. Biologist, Uni- Centro de Ecología. IVIC. nal, Center for Ecology, José D. Medina. Chemist, versidad Central de Venezuela Apartado 21827. Caracas 1020- IVIC, Venezuela. UCV, Venezuela. Doctor in (UCV). Doctor in Agronomy, A. Venezuela. Matilde Gómez. Chemist, Uni- Chemistry, University of University of Hohemheim, e-mail: [email protected] versidad Simón Bolívar, Ven- Laval, Canada. Researcher, Stuttgart Germany. Professor, Guillermina Aguiar. Chemist, ezuela. Associate Research Center for Chemistry, IVIC, UCV and Researcher, Instituto Instituto Pedagógico de Professional, Center for Chem- Venezuela. Venezolano de Investigaciones Caracas, Venezuela. Asso- istry, IVIC, Venezuela. Científicas (IVIC). Address: ciate Research Professio-

OCT 2004, VOL. 29 Nº 10 0378-1844/04/10/579-04 $ 3.00/0 579 RESUMO

O gênero Clusia L. (Clusiaceae) compreende umas 300 es- em base à proporção de alcanos, >90% do total em Clusia pécies que ocorrem desde México e o sul dos EE.UU. até Bolí- rosea, C. orthoneura e C. minor, à presença dos triterpenos α- via e o sul do Brasil. Entre elas se incluem árvores e arbustos, amirina e lupeol em C. multiflora, e de friedelina e taraxerol, hemiepífitas, epífitas e lianas. A análise taxonômica do gênero conjuntamente com C33 e C35 em C. grandiflora e C. se dificulta pela pobre preservação das flores ao ser secadas. schomburgkiana. Os resultados sugerem que a proporção de Este trabalho explora a composição de ceras epicuticulares para alcanos e triterpenóides de ceras epicuticulares tem importância caracterizar espécies mediante marcadores químicos. Se analisou taxonômica e pode ser utilizada para separar espécies ou seções a composição do extrato obtido de seis espécies mediante lavado infragenéricas. da superfície foliar com hexano. As espécies puderam separar-se

help in the identification of Results ecologic and taxonomic groups. The latter is possible Amount of hexane-soluble as the result of the conserva- compounds extracted tive biochemical pathways that lead to wax synthesis The average load of hex- (Bianchi, 1987; Gülz, 1994). ane-soluble compounds (HSC; Extraction and identification Figure 1 for adult leaves) was of wax compounds is rela- relatively higher in C. schom- tively straightforward using burgkiana, C. grandiflora, and volatile low polarity organic C. orthoneura (≥20µg·cm-2), solvents, gas chromatography than in C. multiflora and C. and mass spectrometry. This minor (<20µg·cm-2). On the paper reports on epicuticular abaxial side only C. ortho- wax profiles of five Clusia neura and C. schomburgkiana species in order to test the had values ≥20µg·cm-2. The feasability to a) separate spe- -2 amount of HSC on the cies using the relative propor- Figure 1. Total hexane-soluble compounds (µg·cm ) extracted from the adaxial leaf side of adult tions of alkanes and presence/ surfaces of adult leaves of Clusia species. Under the Abaxial and Adaxial leaves was larger than on the headings bars followed by the same letter are not statistically different. absence of triterpenoids; b) abaxial side in C. rosea and evaluate variations of wax C. grandiflora, while the op- load and composition in rela- Epicuticular waxes were 4ºC/min up to 320ºC and posite occurred in C. schom- tion to leaf age and leaf side, extracted from at least three maintained for 10min. The in- burgkiana and C. orthoneura. and the influence of ecologi- fresh leaf replicates, separat- jector was maintained at In C. multiflora and C. minor cal conditions such as sun ex- ing adaxial and abaxial sides 250ºC and the detector at differences in HSC content on posure. of young and mature, healthy 320ºC. Results are reported as both sides were small. In leaves. Leaf area was mea- amount of extracted waxes young leaves these relation- Material and methods sured before extraction (Licor per unit leaf area, and as rela- ships were similar. No statisti- LI-3100 area meter), and leaf tive proportions as indicated cal differences could be es- Clusia multiflora HBK dry weight was determined by the average percentage of tablished between leaves of (Section Anandrogyne) and after extraction (ventilated chromatogram area for each different ages, or between leaf C. minor L. (Section Retin- oven at 60ºC until constant chemical species. Compound sides, due to the variability in ostemon) shrubs were weight). Leaves were washed identification was done using gravimetric HSC estimations. sampled in a disturbed cloud with 50ml hexane (GC grade) commercial standards, and Therefore, the relationship be- forest (1500m elevation) in for 90sec at room temperature analyzing a subsample in GC/ tween extract weights and the interior coastal range using a pizette. The extract MS Varian 3400CX with a chromatogram areas, as ex- south of Caracas, Venezuela. was evaporated to dryness at Varian Saturn 2000 mass de- pression of total HSCs in- Leaves from C. schom- room temperature. This proce- tector. Mass spectra were jected, was positive and sig- burgkiana Planch. & Triana dure extracts only surface identified using commercial nificant but noisy (data not ex Engler (Section Omphal- hexane-soluble compounds libraries (Wiley Registry of shown) and could not be used anthera; female plant), C. without disturbing the leaf in- Mass Spectral Data and NIST for quantification. grandiflora Splitg. (Section terior. The dry extract was 98 Standard Reference Data- Chlamydoclusia; female weighed (10µg precision), re- base). Composition of the plant), C. orthoneura Standl. dissolved in hexane and sub- Total amount of extracts hexane-soluble compounds (Section Cochlanthera; male sequently injected into a gas per leaf side per species plant) and C. rosea Jacq. chromatograph (Hewlett- were compared using a one- The GC-MS analyses al- (Section Chlamidoclusia; Packard 6890, flame ioniza- way ANOVA (or a Welch lowed the separation of two sterile trees) were sampled tion detector, HP5 column ANOVA for unequal vari- groups of species (Table I). In from trees (one tree per spe- 30m·0.25mm·0.2µm, using He ances); means were com- Group I, HSCs were domi- cies) planted at the Botani- as transport gas). Temperature pared using a Student’s in- nated by alkanes (>90% on cal Garden grounds in was increased at 10ºC/min dividual pair test (SAS, both leaf sides), and it in- Caracas. from 160º to 240ºC, and then 2002). cluded C. orthoneura, C. ro-

580 OCT 2004, VOL. 29 Nº 10 TABLE I LINEAR ALKANES (%) IN CLUSIA SPECIES SEPARATED BY GAS CHROMATOGRAPHY AND IDENTIFIED BY MASS SPECTROMETRY % of compound C20 C24 C25 C26 C27 C28 C29 C30 C31 C32 C33 C35 C36 %T* GROUP I Adaxial 0.8 1.4 1.9 35.5 4.2 48.8 1.3 3.4 0.7 98.1 C. orthoneura Abaxial 0.2 0.4 35.4 4.7 52.7 1.6 3.9 0.2 99.2 Adaxial 0.9 1.1 9.8 4.7 35.3 3.8 31.3 1.9 9.0 0.2 98.0 C. rosea (sun) Abaxial 1.3 0.8 16.3 2.0 48.5 3.5 16.1 0.6 88.9 Adaxial 5.3 3.1 24.9 3.9 38.2 3.4 16.0 0.4 95.4 C. rosea (shade) Abaxial 0.4 8.7 1.3 46.5 4.5 28.8 1.6 91.9 Adaxial 0.7 1.1 32.0 4.5 47.3 2.6 5.6 1.6 95.3 C. minor Abaxial 0.3 0.7 32.8 3.8 49.4 2.6 5.6 0.2 0.3 95.5 GROUP II Adaxial 4.2 4.6 8.3 7.2 9.9 18.8 3.7 9.7 66.4 C. multiflora (male) Abaxial 2.0 0.7 48.5 1.1 36.8 0.5 4.2 93.8 Adaxial 2.2 2.4 3.0 8.4 6.0 10.8 14.5 5.1 9.8 62.2 C. multiflora (fem.) Abaxial 1.0 36.9 1.7 45.9 1.5 7.3 94.2 Adaxial 0.4 0.9 0.7 1.4 2.4 4.7 4.2 28.0 1.4 8.8 9.2 62.0 C. schomburgkiana Abaxial 0.2 0.1 7.9 0.2 53.1 3.8 20.9 0.4 86.7 Adaxial 0.8 7.2 1.4 8.0 2.2 0.8 1.0 30.4 51.8 C. grandiflora Abaxial 1.2 0.6 9.7 1.9 35.8 4.8 33.8 7.1 94.8 * %T: total percent of alkanes. sea, and C. minor. In Group whereas the species of Group parison of the triperpene com- Discussion and Conclusions II alkanes from the adaxial II were characterized by position of the adaxial leaf side constituted <70% of total abundance levels above 20% side within Group II showed The chemical composition HSCs, and it included C. mul- (Table II). The triterpenes distinct patterns. C. multiflora of epicuticular waxes has tiflora, C. schomburgkiana, identified were α-amyrin, was characterized by the pres- been used as an aid to solve and C. grandiflora. friedelin, lupeol, taraxerol and ence of lupeol and α-amyrin, taxonomical problems in Linear alkanes in Group I three unidentifed cholestans. while C. grandiflora and C. higher plants (Salatino et al., were dominated by the com- In addition, the triterpene pre- schomburgkiana were distin- 1989; Mimura et al., 1998). pounds C29 and C31, their cursor squalene was detected guished by the predominance The study of Maffei (1996), ratio being usually >1 in both in small amounts in practi- of the compound friedelin who separated several tribes leaf sides. cally all samples. The com- (Table II). within the Poaceae using the Group II was more hetero- geneous. C29 was the do- TABLE II minant compound in C. mul- TRITERPENES AND SIMILAR COMPOUNDS (%) IDENTIFIED SEPARATED tiflora, particularly on the BY CHROMATOGRAPHY AND IDENTIFIED BY MASS SPECTROMETRY abaxial side. The C29/C31 ratio on the abaxial side was % Compound Squalene Amyrin Lupeol Taraxerol Friedelin Cholestans N-I %T consistently >1 in the male GROUP I plants and <1 in female Adaxial 0.0 C. orthoneura plants. This species was also Abaxial 0.1 0.1 0.2 0.5 characterized by the lack of Adaxial 0.1 0.2 0.4 C35 and the presenc e of C. rosea (sun) Abaxial 0.7 0.7 C24, C25 and C26 on the Adaxial 0.7 0.8 1.5 adaxial side. In C. schom- C. rosea (shade) burgkiana the predominant Abaxial 1.9 2.9 4.8 alkanes were C31, C33 and C. minor Adaxial 0.5 0.5 C35 on both leaf sides. C. Abaxial 0.2 0.2 grandiflora differed in alkane composition depending GROUP II Adaxial 2.3 6.0 18.9 2.9 30.1 on leaf side. On the adaxial C. multiflora (fem) side the most abundant com- Abaxial 1.9 1.9 Adaxial 6.7 4.1 15.2 26.0 pound was C35, whereas on C. multiflora (male) the abaxial sides the com- Abaxial 0.0 pounds C31 and C33 pre- Adaxial 0.3 4.9 10.52 4.3 20.0 C. schomburgkiana dominated, showing similar Abaxial 0.4 0.5 0.75 0.7 2.3 proportions. Adaxial 0.9 1.6 12.38 6.8 21.7 On the adaxial side the C. grandiflora Group I species showed levels Abaxial 0.8 0.8 of triterpenoids below 1.5%, N-I: non-identified

OCT 2004, VOL. 29 Nº 10 581 alkane composition of hexane predominance of alkanes (C. Barthlott W, Wollenweber E tabolism: Biochemistry, Eco- extracts of epicuticular waxes minor, C. orthoneura and C. (1981) Zur Feinstruktur, physiology and Evolution. Chemie und taxonomischen Ecological Studies Vol. 114. is a good example in this di- rosea), while in the rest Signifikanz epicuticularer Springer. Berlin-Heidelberg- rection. However, several au- (Group II) triterpenoids con- Wachse und ähnlicher Sekrete. New York. pp. 296-311. Tropische u. subtropische thors do not encourage this stituted 20-30% of the total Maffei M (1996) Chemotaxonomic Pflanzenwelt 32, Akad. Wiss. type of approach because of HSC detected in the adaxial significance of leaf wax al- Lit. Mainz. F. Steiner Verlag, the predominant alkane mol- leaf side. Triterpene com- kanes in the Gramineae. Stuttgart, 67S. ecules (C29 and C31) are pounds such as lupeol and α- Biochem. Systemat. Ecol. 24: ubiquitous for the whole set amyrin occurred only in C. Barthlott W, Neinhuis C, Cutler D, 53-64. Ditsch F, Meusel I, Theisen I, Marsaioli AJ, Porto ALM, Gon- of higher plants (Barthlott and multiflora, while taraxerol and Wilhelmi H (1998) Classifica- Wollenweber, 1981). Genetic friedelin characterized C. çalves RAC, de Oliveira tion and terminology of plant CMA, Manfio GP, Bittrich V analyses of the biosynthetic grandiflora and C. schom- epicuticular waxes. Bot. J. (1999) The ecosystem of mi- processes involved will pro- burgkiana. Analyses of C. Linnean Soc. 126: 237-260. croorganisms, bees, and vide a better understanding on multiflora waxes suggest that Bianchi G (1987) Chemical genet- Clusia floral resin and oils, how conservative are the bio- it may be possible to identify ics of plant surface lipid bio- from the chemistry point of chemical pathways leading to the sex of the sampled plant synthesis. Gazetta Chimica view. IUPAC. www.iupac.org/ Italiana 117: 707-716. symposia/proceedings/ epicuticular wax components, using the C29/C31 alkane ra- phuket97/marsaioli.html and settle the question of tios from HSC extracted from Bittrich V, Amaral MCE (1996) Flower morphology and polli- Mimura MR, Salatino MLF, specificity of wax composi- the abaxial leaf side. Since nation biology of some Clusia Salatino A, Baumgratz JFA tion spectra (Bianchi, 1987; Clusia is a genus constituted species from the Gran Sabana (1998) Alkanes from foliar Lemieux, 1996; Kunst and predominantly by dioecious (Venezuela). Kew Bulletin 51: epicuticular waxes of Huberia Samuels, 2003). species, this approach opens 681-694. Species: Taxonomic Implica- tions. Biochem. Systemat. One of the problems for interesting possibilities. The Bondada BR, Oosterhuis DM, Ecol. 26: 581-588. using epicuticular wax com- species studied here may be Murohy JB, Kim MS (1996) position as a taxonomic tool considered as intermediate or Effect of water stress on the Neinhuis C, Barthlott W (1997) epicuticular wax composition The tree leaf surface: structure is the variability in the extrac- low wax accumulators on a and ultrastructure of cotton and function. In Renneberg H, tion power of different or- per unit leaf area, compared (Gossypium hirsutum L.) leaf, Esrich W, Ziegler H (Eds.) ganic solvents reported in the to species from other environ- bract, and boll. Environ. Exp. Trees- Contributions to mod- literature, and the use of fresh ments (Oliveira and Salatino, Bot. 36: 61-69. ern tree physiology. Backhuys. Leiden, Netherland. pp. 3-18. or dried leaves (Stammiti et 2000). We did not detect dif- Cuesta-Rubio O, Frontana-Uribeb al., 1996). Differences in pen- ferences in the amount or BA, Ramírez-Apanb T, Cárde- Neinhuis C, Koch K, Barthlott W etration depth of the solvent composition of alkanes be- nas J (2002) Polyisoprenylated (2001) Movement and regen- Benzophenones in Cuban Pro- eration of epicuticular waxes into the cuticula results in un- tween sun and shade leaves polis; Biological Activity of through plant cuticles. Planta certainty about the actual lo- of C. rosea; however, the Nemorosone. Z. Naturforsch. 213: 427-434. calization of the extracted shade samples of this species 57c: 372-378. Oliveira AFM, Salatino A (2000) substances. Jetter and Schäffer were richer in triterpenoids. Engler A (1925) Clusia. In Engler Major constituents of the fo- (2001) showed that in Prunus These results may be associ- A, Prantl K (Eds.) Die liar epicuticular waxes of spe- laurocerasus triterpenoids are ated with the mild conditions Natürliche Pflanzenfamilien. cies from the Caatinga and 2nd ed. Vol. 21: 202. Cerrado. Z. Naturforsch. 55c: restricted to the intracuticular experienced during develop- 688-692. matrix, while the alyphatic ment of the harvested leaves. Franco AC, Ball E, Lüttge U (1990) Patterns of gas ex- Pipoly IIIJ, Kearns DM, Berry PE compounds were the only The measured differences in (1998) Clusia L., Sp. Pl. 509. truly epicuticular components. composition and proportions change and organic acid oscil- lations in tropical trees of the 1753. In Steyermark JA, However, triterpenes could be of epicuticular waxes within genus Clusia. Oecologia 85: Berry PE, Holst BK, transported to the superficial this small set of species, repre- 108-114. Yatskievych K (Eds.) Flora of wax layer, if the mechanism senting large sections within the Venezuelan Guayana. Vol. Gülz PG (1994) Epicuticular 4. Missouri Botanical Garden. for movement and regenera- the genus, suggest the poten- waxes in the evolution of the St. Louis. MO, USA. tion of epicuticular waxes tial chimio-taxonomic value of plant kingdom. J. Plant Physiol. 143: 453-464. Salatino MLF, Salatino A, through plant cuticles de- this approach, which deserves Menezes NL, Silva RM scribed by Neinhuis et al. further investigation. Jetter R, Schäffer S (2001) Chemi- (1989) Alkanes of Foliar Epi- (2001) is of general occur- cal Composition of the cuticular Waxes of Prunus laurocerasus Leaf Sur- Velloziaceae. Phytochemistry rence in higher plants. These ACKNOWLEDGEMENTS authors concluded that wax face. Dynamic Changes of the 28: 1105-1114. Epicuticular Wax Film during ® moves through the cuticle in a Silvia Llamozas (formerly Leaf Development. Plant SAS (2002) JMP The Statistical process analogous to steam at the Fundación Instituto Physiol. 126: 1725-1737. Discovery Software. SAS In- stitute Inc. Cary, NC, USA. distillation. 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